Biosynthesis of endotubin: an apical early endosomal glycoprotein from developing rat intestinal epithelial cells.
Endosomes are the site of sorting of internalized receptors and ligands in all cell types and, in polarized cells, the apical endosomal compartment is involved in the selective transepithelial transport of immunoglobulins and growth factors. The biochemical composition of this specialized compartment remains largely unresolved. We have characterized a glycoprotein, called endotubin, that is located in the apical endosomal tubules of developing rat intestinal epithelial cells. A monoclonal antibody against endotubin recognizes a broad band of 55-60kDa, which upon isoelectric focusing can be resolved into two bands, and a faint band of 140kDa. Metabolic labelling followed by immunoprecipitation indicates that endotubin is synthesized as a 140kDa precursor that is cleaved to the 55-60kDa forms. High pH washing of endosomal membranes removes the 55-60kDa forms from the membrane, whereas the high-molecular-mass form remains membrane associated and appears to be an integral membrane protein. Immunoblotting with a polyclonal antibody against the putative cytoplasmic tail of the protein identifies a 140kDa band and a band of 74kDa, presumably the cleavage product. Immunoprecipitation with antibodies against the 55-60kDa form results in coprecipitation of a 74kDa protein, and immunoprecipitation with antibody against the 74kDa protein results in coprecipitation of the 55-60kDa form. Epitope mapping of the monoclonal antibody binding site supports a proposed type I membrane protein orientation. We propose that endotubin is proteolytically processed into a heterodimer with the 55-60kDa fragment remaining membrane-associated through a non-covalent association with the membrane-bound 74kDa portion of the molecule.
Allen
K
K
Department of Cell Biology and Anatomy, Steele Memorial Children's Research Center, University of Arizona, Tucson, AZ 85724, USA.
Gokay
K E
KE
Thomas
M A
MA
Speelman
B A
BA
Wilson
J M
JM
D000818:Animals; D000831:Animals, Newborn; D000911:Antibodies, Monoclonal / Q000276:immunology; D011992:Endosomes / Q000737:chemistry / Q000378:metabolism*; D004847:Epithelial Cells / Q000378:metabolism; D018604:Epitope Mapping; D006026:Glycoside Hydrolases / Q000378:metabolism; D007082:Ileum; D007158:Immunologic Techniques; D007413:Intestinal Mucosa / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000737:chemistry; D008970:Molecular Weight; D017433:Protein Structure, Secondary; D051381:Rats; D017386:Sequence Homology, Amino Acid
D000911:Antibodies, Monoclonal; D008562:Membrane Glycoproteins; D006026:Glycoside Hydrolases